Means for biologically controlling cryptogamic plant diseases

ABSTRACT

The invention concerns the application of compositions of micro-organisms in biological control of vin crptogamic diseases. Said composition comprises a mixture of at least one bacterium and at least one yeast, the bacterium or bacteria and the yeast(s) being non-toxic for the plant. The invention also concerns bacterial and yeast strains, as well as biofungicide formulations containing an efficient amount of at least one composition of micro-organisms including in mixture at least one bacterium and one yeast, the bacterium or bacteria and the yeast(s) being non-toxic for the plant, and a composition of filamentous fungi, in particular of the genus  Pichia, Pythium, Trichoderma, Gliocladium, Ampelomyces, Talaromyces, Epicococcum , combined with an inert carrier. The invention is useful for treating cryptogamic plant diseases, in particular crop plants and vine.

The invention is aimed at means, namely compositions and methods, forbiologically controlling cryptogamic plant diseases, i.e. caused bypathogenic fungi.

Biological control, i.e. the use of living microorganisms to combatplant diseases, arouses much interest given the growing problems posedby the use of chemical fungicides in agriculture. Polluted water tables,contaminated soils, and the resistance of pathogenic fungi to thefungicides are all challenges to be overcome by modern agriculture.

In the field of viticulture for example, it is necessary to produce everhigher quality wines and to reduce the pollution caused by the intensiveuse of chemicals.

Bacteria, such as Bacillus thuringiensis, or fungi, such as Beauveria orEndothia have already been proposed as biological control means.

Now, the inventors have been able to develop highly effective non-toxicmeans by using microorganisms in a mixture to control cryptogamic vinediseases. Such mixtures not only act in synergy in the desiredbiological control, but also advantageously have an eliciting effect.

Moreover, it has been observed that particular mixtures ofmicroorganisms were particularly effective for the treatment ofcryptogamic plant diseases in general.

The purpose of the invention is therefore the use of microorganisms inthe biological control of cryptogamic vine diseases.

It also aims to provide new compositions of microorganisms and their useas means for biologically controlling cryptogamic plant diseases, morespecifically cryptogamic diseases in crop plants.

The use of compositions of microorganisms in the biological control ofcryptogamic vine diseases is characterized in that it comprises in amixture at least one bacterium and at least one yeast, the bacterium orbacteria and the yeast or yeasts being non-toxic to plants.

Advantageously, the bacterium or bacteria are chosen from the Bacillus,Pseudomonas, Serratia or Streptomiyces groups.

This bacterium or bacteria is in particular from the Bacillus megateriumspecies, and in particular from the strain deposited on the 20 Jun. 2002at the CNCM, 25 rue du Dr. Roux, 75724 Paris Cedex 15, under No. I-2897and bacterium or bacteria from the Bacillus subtilis species depositedon the 20^(th) June at the same institution under No. I-2940.

The yeast or yeasts of the compositions of the invention areadvantageously of the Debaryomyces or Pichia genus. In particular it isthe yeast of the Debaryomyces genus deposited on the 20 June at the CNCMunder No. I-2896.

In particular the invention relates to, as newly isolated products, theBacillus megaterium (BP B-01) strain deposited on the 20 Jun. 2002 atthe CNCM, 25 rue du Dr. Roux, 75724 Paris Cedex 15, under No. I-2897 andthe Bacillus subtilis (BP B-10) strain deposited at the CNCM on the 20Jun. 2002 under No. I-2940.

The invention is also relates to, as a newly isolated product, the yeastof the Debaryomyces (BP Y-01) genus deposited on the 20 Jun. 2002 at theCNCM under No. I-2896.

The strains of bacteria and yeast of the invention are advantageouslyselected from microorganisms from the soil or taken from the plant to betreated. Therefore they do not disturb the biological balance of theecosystem because they originate from it.

The compositions of these microorganisms allow treatment of the mainplant fungus diseases and are therefore particularly suitable fordeveloping biofungicide preparations.

Preparations of the invention are characterized in that they contain aneffective quantity of at least one composition of microorganismscomprising in a mixture at least one bacteria and at least one yeast,the bacterium or bacteria and the yeast or yeasts being non-toxic to theplant, as well as a composition of filamentous fungi, in particular ofthe Pichia; Pythium, Trichoderma, Gliocladium, Ampelomyces, Talaromyces,Epicococcum genus in combination with an inert vehicle.

The vehicle used is in particular able to ensure good dispersion andadhesion of the microorganisms on the aerial part of the plant.

Preferred biofungicide preparations are characterized in that theycontain an effective quantity of the bacterial strains and of the strainof yeast deposited at the CNCM, as defined above. Preferably, thebiofungicide preparations of the invention are characterized byconcentrations of yeasts of 0.5 to 0.75×10¹⁰ and of bacteria of 0.30 to0.50×10¹⁰.

These preparations have a broad spectrum of effectiveness againstpathogenic fungi and are advantageously used for the treatment ofcryptogamic diseases in plants, in particular in crop plants, inparticular in the aerial parts for example vine, tomato, strawberry,wheat, potato, tobacco, sugar cane, corn, rice, fruit-trees, in tubersfor example potatoes or beet.

The effectiveness was also noted in particular in market-garden plants.

They are used for example in the treatment of powdery mildew, bluemould, and Botrytis of the vine, as well as wood diseases (esca oreutypa).

These preparations also have the advantage of having an eliciting effectwhich causes activation of the natural defences of the plant againstaggressors of fungal origin.

The treatments are carried out in particular by spraying on the aerialparts with preparations containing the bacterium or bacteria and theyeast or yeasts, with quantities which are adjustable according to thepathogen pressure. Suitable preparations contain for example saidbacteria and yeasts in a ratio of approximately 50/50%.

It will be noted that the application of these treatments does notrequire any change of equipment on the part of the user.

According to another provision of the invention, the treatment iscompleted, if appropriate, using a composition of fungi, morespecifically of filamentous fungi, in particular of the Pythium,Trichoderma, Gliocladium, Ampelomryces, Talaromryces, Epicococcum genus.

After the application, the strains of the preparation remain on theplant for a few days only before dying and decomposing. Their presencein the soil is not greater than a level normally encountered and isnon-existent below thirty centimetres in depth.

These mixtures of microorganisms therefore do not produce any new formof pollution.

The invention also aims to take advantage of the eliciting effect of themixtures of microorganisms of the invention to produce compounds ofinterest, such as resveratrol, a molecule having in particular aprotective effect on the cardiac system, or also pterostilbene, inhigher quantities than those produced by the vine after elicitation suchas produced by Botrytis.

The invention is thus aimed at a method for the production ofresveratrol and/or pterostilbene, comprising the application to a plant,in particular vines, of a composition of microorganisms as definedabove, and the recovery of resveratrol and/or pterostilbene for exampleby extraction.

Resveratrol can be used for the production of medicaments in particularfor cardiovascular purposes, in cosmetology or as a nutriceutical.Pterostilbene can be used as an antifungal.

Other characteristics and advantages of the invention will becomeapparent from the following examples given by way of illustration of theinvention and with reference to the single FIGURE which presents theproduction of resveratrol with different combinations of bacteria and/oryeasts and/or fungi.

EXAMPLE I Isolation and Culture of the Strain of Bacteria BP B01 and ofYeast BP Y01

The isolation takes place as follows:

The two microorganisms are cultured on a solid PDA (potato dextroseagar) nutrient medium. The two microorganisms are seeded separately in20 litre fermenters containing PDB (potato dextrose broth). The yeastsare cultured at 28° C. and the bacteria at 37° C. The microorganismsthus prepared are centrifuged and the pellet of bacteria and yeasts isrecovered separately in sterile distilled water. When the plant istreated, the two microorganisms are mixed. The vineyard undergoes threetreatments per month, from April to August, thus a total of 10 to 12treatments with the mixture of the invention for the season. The productprepared by fermentation is used for the whole season, each litrecontaining from 0.5-0.75×10¹⁰ for the yeasts and 0.30-0.50×10¹⁰ for thebacteria, at a rate of 1 litre per vine trunk.

Characterization of the Genomic DNA of BP B01 and BP B10

The isolation of the genomic DNA of BP B01 and of BP B10, as well as theamplification of their ITS region, between the 16S and 23S genes wascarried out according to Chen. W, Schneider R. W, and Hoy J. W. 1992,Phytopathology, 82, p. 1234-1244. The corresponding sequences SEQ ID No.1 and SEQ ID No. 3 were deposited at GENBANK (access No.s AY 125961 andAY 157575 respectively).

The yeast BP Y01 was isolated from grapes from different vineyards ofthe Burgundy region.

The genomic DNA of BP Y01 was isolated and the ITS region, between the18S and 28S genes was amplified according to the above methods.

The ITS1 fragment is located between the 18S and 5,8 S genes, while ITS2is located between the 5,8 S and 28 S genes. The sequence SEQ ID No. 2was deposited at GENBANK (access No. AY 125962).

Results of Biological Control on Chardonnay Grapes in 1999 and 2000 atthe Centre Expérimental de Marsannay-La-Côte, France (Université deBourgogne) Year: 1999 Results Number of trunks 450 Non-treated controls 10 Number of treatments  15 Type of inoculum Bacteria + yeastsProtection 39 diseased trunks (blue mould), O Botrytis Controls Bluemould then Botrytis on all the trunks, chemical intervention Year: 2000Results Number of trunks 241 Number of treatments  14 Type of inoculumBacteria + yeasts Protection 27 diseased trunks (blue mould), O BotrytisControls Blue mould then Botrytis on all the trunks

Results of Biological Control on Pinot-Noir Grapes in 2000 and 2001 atthe Centre Expérimental de Marsannay-La-Côte, France (Université deBourgogne) Year: 2000 Results Number of trunks 120 Non-treated controls 13 Number of treatments  14 Type of inoculum Bacteria + yeasts ControlsBlue mould then Botrytis on all the trunks Protection 11 diseased trunks(blue mould), O Botrytis Year: 2001 Results Number of trunks 614 Numberof treatments  13 Chemical treatment  02 Non-treated controls  13 Typeof inoculum Bacteria + yeasts Controls Blue mould + Botrytis on all thetrunks Protection 53 diseased trunks (blue mould), a few bunchesinfected with Botrytis Yield 1.18 kg of grapes per trunk instead of1.2-1.5 kg

Generally, in addition to the fungicide and eliciting effect of thepreparations used, an improvement in the quality of the grape harvest isnoted. In particular an increase in the concentration of sugar whichallows chaptalization to be reduced or avoided is observed. Moreover,the level of resveratrol is increased. Finally, the levels of tannin andanthocyanins increase, which gives the wine greater fullness andbrighter colour.

The results relating to the increase of resveratrol are illustrated bythe histogram of the single FIGURE which gives in ug/gm of fresh weightthe production of resveratrol on the 3^(rd) day after additionrespectively of: BP Y01 (yeast deposited at the CNCM); BP B01 (bacteriadeposited at the CNCM); BC 03 (botrytis cinerea); BP Y01+B C03; BPB 01+BC03; BP Y01+BP B01; BP B01+BP Y01+B C03.

Examination of this histogram shows that the formation of resveratrolincreases when the yeast/bacteria mixture of the invention is added tothe Botrytis fungus.

Taking into account all of these properties, the compositions used asfungicide preparations, according to the invention, constitute aparticularly satisfactory response to the fungicide problems whichgrowers, in particular wine growers are facing and to the need to reducepollution caused by agriculture by constituting substitutes to chemicalproducts.

1. Application of compositions of microorganisms for biologically controlling the cryptogamic vine diseases, characterized in that it comprises in a mixture at least one bacterium and at least one yeast, the bacterium or bacteria and the yeast or yeasts being non-toxic to the plant.
 2. Application according to claim 1, characterized in that the bacterium or bacteria are chosen from the Bacillus, Pseudomonas, Serratia or Streptomyces genera.
 3. Application according to claim 2, characterized in that the bacterium or bacteria are from the Bacillus megaterium species and/or from the Bacillus subtilis species.
 4. Application according to any one of claims 1 to 3, characterized in that the yeast or yeasts are of the Debaryomyces or Pichia genus.
 5. Bacterial strain, characterized in that it is the Bacillus megaterium (BP B-01) strain deposited on the 20 Jun. 2002 at the CNCM under No. I-2897.
 6. Bacterial strain, characterized in that it is the Bacillus subtilis (BP B-10) strain deposited on the 20 Jun. 2002 at the CNCM under No. I-2940.
 7. Yeast strain, characterized in that it is the yeast of the Debaryomyces (BP Y-01) genus deposited on the 20 Jun. 2002 at the CNCM under no. I-2896.
 8. Biofungicide preparation, characterized in that it contains an effective quantity of at least one composition of microorganisms comprising in a mixture at least one bacterium and at least one yeast, the bacterium or bacteria and the yeast or yeasts being non-toxic to the plant, as well as a composition of filamentous fungi, in particular of the Pichia, Pythium, Trichoderma, Gliocladium, Ampelomyces, Talaromyces, Epicococcum genus in combination with an inert vehicle.
 9. Biofungicide preparation according to claim 8, characterized by concentrations of yeast of 0.5 to 0.75×10¹⁰ and of bacteria of 0.30 to 0.50×10¹⁰.
 10. Biofungicide preparation, characterized in that it contains an effective quantity of a mixture of strains according to claims 5 to
 7. 11. Application of a preparation according to one of claims 8 to 10, for the treatment of cryptogamic diseases in plants, in particular for crop plants, in particular for the aerial parts for example vine, tomato, strawberry, wheat, tobacco, sugar cane, corn, rice, fruit-trees, tubers for example potatoes or beet.
 12. Application of a preparation according to one of claims 8 to 10 for the treatment of powdery mildew, blue mould or Botrytis of the vine, and eutypa and esca.
 13. Application according to claim 11 or 12, characterized in that it comprises a treatment in particular by spraying with the bacterium or bacteria and said yeast or yeasts, according to the pathogen pressure, in particular according to a ratio of approximately 50/50%.
 14. Application according to any one of claims 11 to 13, characterized in that it also comprises a treatment using a composition of filamentous fungi, in particular of the Pichia, Pythium, Trichoderma, Gliocladium, Ampelomyces, Talaromyces, Epicococcum genus.
 15. Application of the biofungicide preparation according to any one of claims 8 to 10 as eliciting agent for the production of substances of interest by the treated plant, in particular of resveratrol. 